To clone and characterize Hypsibius henanensis GATA binding protein 3 (HhGATA3) and to investigate its function in radiation tolerance.

The coding sequence of HhGATA3 was amplified from H.henanensis cDNA by PCR, and the protein was analyzed using bioinformatics tools. The subcellular aggregation of HhGATA3 was observed by immunofluorescence. The radioprotective capacity was evaluated via alkaline comet assay and phosphorylated histone H2AX (γH2AX) foci formation in human cells expressing HhGATA3. The knockdown efficiency of small interfering RNA (siRNA) was verified by reverse transcription real-time quantitative PCR (qPCR) in H.henanensis, and the post-irradiation survival rate was measured.

The HhGATA3 gene was cloned. HhGATA3 protein formed phase-separated droplets within cells. Comet and γH2AX foci assays suggested that HeLa cells expressing HhGATA3 had their DNA damage significantly mitigated after X-ray irradiation (P<0.0001). Knockdown of HhGATA3 in H. henanensis markedly decreased the post-irradiation survival rate to approximately 40% (P<0.001).

HhGATA3 plays a critical role in the radiation tolerance of H.henanensis. In human HeLa cells, HhGATA3 can undergo phase separation and mitigate radiation-induced DNA damage.

To establish a method for isolation and short-term culture of storage cells of Hypsibius henanensis, characterize their features, and evaluate their radiation tolerance so as to provide an in vitro platform for investigating stress-resistance mechanisms of tardigrades.

Laboratory-cultured H.henanensis was used as the subject. Storage cells were mechanically isolated. The osmotic pressure of the tardigrade homogenate was measured to optimize the culture conditions. The isolated cells were maintained in a short-term culture using modified Grace’s medium. The cell viability was determined via trypan blue exclusion and calcein acetoxymethyl ester (Calcein-AM) and propidium iodide (PI) double staining. The morphology and components of these cells were characterized by fluorescence staining. Radiation tolerance was evaluated by comparing the survival rate of storage cells with that of HeLa cells after exposure to 500 Gy of X-ray irradiation.

High-purity storage cells were isolated, which were round or polygonal in shape with an average diameter of (7.50±0.15) µm. These cells had intact cell membranes and contained abundant cytoplasmic neutral lipid droplets, with mitochondria evenly distributed. The optimized culture system could keep cell viability stable for 7 d. After 500 Gy X-ray irradiation, the survival rate of storage cells was (76.30±17.32)%, which was not significantly different from that of the non-irradiated group (74.71±14.74%) (P>0.05), unlike HeLa cells whose survival rate decreased significantly (P<0.01).

A method for isolation and short-term culture of storage cells from H.henanensis has been established. These storage cells are abundant in lipid reserves and capable of radiation tolerance in vitro. These findings can help construct cell models and study the stress-resistance mechanisms of tardigrades.